N-(pyrimidinyl)-aspartic acid analogs as interleukin-1β converting enzyme inhibitors

ABSTRACT

Disclosed are compounds, compositions and methods for inhibiting interleukin-1β (1L-β) protease activity. The compounds, N-(pyrimidinyl)-aspartic acid α-substituted methyl ketones and aspartic acid aldehydes, have the formula (I) set out herein. 
     These compounds are inhibitors of 1β-converting enzyme and as such are useful whenever such inhibition is desired. For example, they may be used as research tools in pharmacological, diagnostic and related studies and in the treatment of diseases in mammals in which IL-β protease activity is implicated.

This application is a continuation-in-part of application Ser. No.08/221,712, filed Mar. 31, 1994, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a series of novel aspartic acid analogs whichexhibit selective in vitro and in vivo inhibition of interleukin-1βconverting enzyme, to compositions containing the novel aspartic acidanalogs and to methods for therapeutic utility. More particularly, theinterleukin-1β converting enzyme inhibitors described in this inventioncomprise novel N-(pyrimidinyl)-aspartic acid aldehydes and α-substitutedmethyl ketones which possess particular utility in the treatment ofinflammatory and immune-based diseases of lung, central nervous system,kidneys, joints, eyes, ears, skin, gastrointestinal tract, urogenitalsystem and connective tissues.

2. Reported Developments

Interleukin-1β (IL-1β) protease (also known as interleukin-1β convertingenzyme or ICE) is the enzyme responsible for processing of thebiologically inactive 31 kD precursor IL-1β to the biologically active17 kD form (Kostura, M. J.; Tocci, M. J.; Limjuco, G.; Chin, J.;Cameron, P.; Hillman, A. G.; Chartrain, N. A.; Schmidt, J. A., Proc.Nat. Acad. Sci., (1989), 86, 5227-5231 and Black, R. A.; Kronheim, S.R.; Sleath, P. R., FEBS Let., (1989), 247, 386-391). In addition toacting as one of the body's early responses to injury and infection,IL-1β has also been proposed to act as a mediator of a wide variety ofdiseases, including rheumatoid arthritis, osteoarthritis, inflammatorybowel disease, sepsis, acute and chronic myelogenous leukemia andosteoporosis (Dinarello, C. A.; Wolff, S. M., New Engl. J. Med., (1993),328, 106). A naturally occurring IL-1β receptor antagonist has been usedto demonstrate the intermediacy of IL-1β in a number of human diseasesand animal models (Hannum, C. H.; Wilcox, C. J.; Arend, W. P.; Joslin,G. G.; Dripps, D. J.; Heimdal, P. L.; Armes, L. G.; Sommer, A.;Eisenberg, S. P.; Thompson, R. C., Nature, (1990), 343, 336-340;Eisenberg, S. P.; Evans, R. J.; Arend, W. P.; Verderber, E.; Brewer, M.T.; Hannum, C. H.; Thompson, R. C., Nature (1990), 343, 341-346;Ohlsson, K.; Bjork, P.; Bergenfeldt, M.; Hageman, R.; Thompson, R. C.,Nature, (1990), 348, 550-552; Wakabayashi, G., FASEB, (1991), 338-343;Pacifici, R.; et al. Proc. Natl. Acad. Sci. (1989), 86, 2398-2402 andYamamoto, I.; et al. Cancer Rsh (1989), 49, 4242-4246). The specificrole of IL-1β in inflammation and immunomodulation is supported by therecent observation that the cowpox virus employs an inhibitor of ICE tosuppress the inflammatory response of its host (Ray, C. A. et al, Cell,(1992), 69,597-604).

In summary, the utility of ICE inhibitors in modifying certain IL-1βmediated disease states has been suggested and demonstrated in vivo byseveral workers in the field. The following review of the current stateof the art in ICE research further supports such utility of ICEinhibitors:

1) WO 9309135, published May 11, 1993, teaches that peptide-basedaspartic acid arylacyloxy-and aryoxymethyl ketones are potent inhibitorsof ICE in vitro. These compounds also specifically inhibited ICE in thewhole cell (in vivo) by their ability to inhibit the formation of matureIL-1β in whole cells. These ICE inhibitors also demonstrated utility inreducing fever and inflammation/swelling in rats.

2) Patients with Lyme disease sometimes develop Lyme arthritis. B.burgdorferi, the causative agent of Lyme disease, is a potent inducer ofIL-1 synthesis by mononuclear cells. Miller et al. (Miller, L. C.;Lynch, E. A. Isa, S.; Logan, J. W.; Dinarello, C. A.; and Steere, A. C.,"Balance of synovial fluid IL-1β and IL-1 Receptor Antagonist andRecovery from Lyme arthritis", Lancet (1993) 341; 146-148) showed thatin patients who recovered quickly from Lyme Arthritis, the balance insynovial fluid of IL-1 -beta and IL-1 ra was in favor of IL-ra. When thebalance was shifted in favor of IL-1β, it took significantly longer forthe disease to resolve. The conclusion was that the excess IL-1rablocked the effects of the IL-1β in the patients studied.

3) IL-1 is present in affected tissues in ulcerative colitis in humans.In animal models of the disease, IL-1β levels correlate with diseaseseverity. In the model, administration of 1L-1ra reduced tissue necrosisand the number of inflammatory cells in the colon.

See, Cominelli, F.; Nast, C. C.; Clark, B. D.; Schindler, R., Llerena,R.; Eysselein, V. E.; Thompson, R. C.; and Dinarello, C. A.;"lnterleukin-1 Gene Expression, Synthesis, and Effect of Specific IL-1Receptor Blockade in Rabbit Immune Complex Colitis" J. Clin.Investigations (1990) Vol. 86, pp, 972-980.

4) IL-1ra supresses joint swelling in the PG-APS model of arthritis inrats. See Schwab, J. H.; Anderle, S. K.; Brown, R. R.; Dalldorf, F. G.and Thompson, R. C., "Pro- and Anti-Inflammatory Roles of Interelukin-1in Recurrence of Bacterial Cell Wall-Induced Arthritis in Rats". Infect.Immun. (1991 ) 59; 4436-4442.

5) IL-1ra shows efficacy in an small open-label human RheumatoidArthritis trial. See, Lebsack, M. E.; Paul, C. C.; Bloedow, C. C.;Burch, F. X.; Sack, M. A.; Chase, W., and Catalano, M. A. "SubcutaneousIL-1 Receptor Antagonist in Patients with Rheumatoid Arthritis", Arth.Rheum. (1991 ) 34; 545.

6) IL-1 appears to be an autocrine growth factor for the proliferationof chronic myelogenous leukemia cells. Both IL-1ra and sIL-1R inhibitcolony growth in cells removed from leukemia patients.

See, Estrov, Z.; Kurzrock, R.; Wetzler, M.; Kantarjian, H.; Blake, M.;Harris, D.; Gutterman, J. U.; and Talpaz, M., "Supression of ChronicMyelogenous Leukemia Colony Growth by Interleukin-1 (IL-1) ReceptorAntagonist and Soluble IL-1 Receptors: a Novel Application forInhibitors of IL-1 Activity". Blood (1991 ) 78; 1476-1484.

7) As in 6) above, but for acute myelogenous leukemia rather thanchronic myelogenous leukemia.

See, Estrov, Z.; Kurzrock, R.; Estey, E.; Wetzler, M.; Ferrajoli, A.;Harris, D.; Blake, M.; Guttermann, J. U.; and Talpaz, M. "Inhibition ofAcute Myelogenous Leukemia Blast Proliferation by Interleukin-1 (IL-1)Receptor Antagonist and Soluble IL-1 Receptors". (1992) Blood 79;1938-1945.

Accordingly, disease states in which the ICE inhibitors of Formula I maybe useful as therapeutic agents include, but are not limited to,infectious diseases where active infection exists at any body site, suchas meningitis and salpingitis; complications of infections includingseptic shock, disseminated intravascular coagulation, and/or adultrespiratory distress syndrome; acute or chronic inflammation due toantigen, antibody, and/or complement deposition; inflammatory conditionsincluding arthritis, cholangitis, colitis, encephalitis, endocarditis,glomerulonephritis, hepatitis, myocarditis, pancreatitis, pericarditis,reperfusion injury and vasculitis. Immune-based diseases which may beresponsive to ICE inhibitors of Formula I include but are not limited toconditions involving T-cells and/or macrophages such as acute anddelayed hypersensitivity, graft rejection, andgraft-versus-host-disease; auto-immune diseases including Type Idiabetes mellitus and multiple sclerosis.

All of the inhibitors of ICE described in the art known to Applicantsare peptide-based, taking advantage of the substrate specificity of theenzyme. We describe in this invention non-peptide based inhibitors ofICE, specifically where the pyrimidine serves as a recognition surrogatefor the P2 and P3 amino acids which up until now had to be present toyield a potent ICE inhibitor (see structure 1). One well-known advantageof non-peptide inhibitors versus their peptide counterpart is that invivo metabolism and excretion of such non-peptidic agents to greatlyattenuated, thereby leading to enhanced bioavailability of thesecompounds in animals and humans (Humphrey, M. J. and Ringrose, P. S.,"Peptides and Related Drugs: A Review of Their Absorption, Metabolism,and Excretion", Drug Metabolism Reviews, (1986), 17, 283-310. AlsoPlattner, J. J. and Norbeck, D. W. "Obstacles to Drug Development fromPeptide Leads", Drug Discovery Technologies, (1990), Chapter 5, 92-126,C. R. Clark and W. H. Moos, eds.; Horwood: Chichester, U.K. ##STR1##

It should be noted that the pyrimidine-based trifluoromethyl ketones(structure 2) were recently described as inhibitors of the sereneprotease, elastase. Since ICE is a cysteine protease and it is known inthe prior art that trifluoromethyl ketones are rather poor inhibitors ofcysteine proteases (See, Imperialia, B. and Ables, R. H., Biochemistry.(1986), 25, 3760-7), it is expected that the pyrimidines of FIG. 2 wouldnot be inhibitors of ICE. Also, it is known that ICE requires theaspartic acid side chain (--CH₂ COOH) at PI. Pyrimidines which inhibitelastase (FIG. 2) contain the valine side chain (--CHMe₂). In addition,as will be shown later, the pyrimidine-based ICE inhibitors(Structure 1) described in this invention do not inhibit human leucocyteelastase and hence are exquisitely selective for ICE and distinct fromthe known elastase inhibitors. ##STR2##

SUMMARY OF THE INVENTION

Compounds of formula I have been found to be potent inhibitors ofinterleukin-1β converting enzyme (ICE). Compounds of formula I areuseful in the treatment of diseases including inflammation in lung,central nervous system, kidney, joints, eyes, ears, skin,gastrointestinal tract and connective tissues.

According to the present invention, there is provided a compound of theformula (I) or a pharmaceutically acceptable salt thereof. ##STR3##

R₅ is H or deuterium;

R₆ is OR₈ or NHOH

where R₈ is independently H, alkyl, or aralkyl

R₃ and R₄ =independently H, alkyl or aralkyl

R₂ ═H, alkyl, --(CH₂)₀₋₄ -cycloalkyl, ##STR4## aryl, heteroaryl,aralkyl, heteroaralkyl, --(CH₂)₂₋₄ --R₁₀ ;

where n=1-3;

where R₁₀ =alkoxy, CH₂ F, CHF₂, CF₃, CF₂ CF₃, OH, COOR₁₁, CONR₉ R₁₁, orNR_(g) R₁₁ ;

where R₉ is independently H, alkyl, aryl, aralkyl, heteroaryl,heteroaralkyl,

    --CH.sub.2 CH.sub.2 O-alkyl and C(O)--R.sub.12 ;

where R₁₁ is independently H, alkyl, aryl, aralkyl, heteroaryl andheteroaralkyl;

and when R₉ and R₁₁ are taken together, they can equal a five, six orseven membered ring of the type: ##STR5## where n=1-3 and m=0-1; and R₁₂is alkyl, aryl, aralkyl, heteroaryl and heteroaralkyl;

R₇ ═H, CH₂ F, CHR₁₃ O(CO)₀₋₁ -aryl, CHR₁₃ OP(O)(R₁₄)R.sub.(15) ##STR6##wherein: R₁₃ =H or alkyl

R₁₄ =H, alkyl or aryl

R₁₅ =H, alkyl or aryl

R₁₆ =H, alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl

R₁₇ =H, alkyl, CF₃, CF₂ CF₃, aryl, heteroaryl, aralkyl, heteroaralkyl,COOR₁₁, or CONR₉ R₁₁

R₁₈ =H, alkyl, CF₃, CF₂ CF₃, aryl, heteroaryl, aralkyl, heteroaralkyl

R₁ is defined as:

R₁₉ -R₂, R₁₉ -R₂₀, R₁₉ -R₂₁, R₁₉ -NR₉ R₁₁

where R₁₉ =(CR₃ R₄)--₀₋₄ ;

R₂₀ = ##STR7## where X=O, S, NR₉ R₂₁ = ##STR8## where n=1-3; R₂₂O--where R₂₂ --alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, R₁₉-cycloalkyl, R₁₉ -R₂₁, R₂₃ -R₁₀, R₂₃ -R₂₀ ;

wherein R₂₃ =(CR₃ R₄)--₂₋₄ ;

R₉ R₁₁ N-- and R₂₄ R₁₁ N--

where R₂₄ =R₁₉ -cycloalkyl, R₁₉ -R₂₁, R₂₃ -R₁₀, R₂₃ -R₂₀, CR₃ R₄ COOR₁₁and CR₃ CR₄ CONR₉ R₁₁ ;

As used herein, the term "pharmaceutically acceptable salts" include theacid and base addition salts.

The term "acid addition salts" refers to those salts which retain thebiological effectiveness and properties of the free bases and which arenot biologically or otherwise undesirable, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

The term "base addition salts" include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Particularlypreferred are the ammonium, potassium, sodium, calcium and magnesiumsalts derived from pharmaceutically acceptable organic non-toxic basesinclude salts of primary, secondary, and tertiary amines, substitutedamines including naturally occurring substituted amines, cyclic aminesand basic ion exchange resins, such as isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, ethanolamine,2-dimethylaminoethanol, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaines,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline and caffeine.

As employed above and throughout the disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings:

"Alkyl" is defined as a saturated aliphatic hydrocarbon which may beeither straight- or branched-chain. Preferred groups have no more thanabout 12 carbon atoms and may be methyl, ethyl, propyl, and so on andstructural isomers of propyl, butyl, pentyl, hexyl, octyl, nonyl, decyl,undecyl, dodecyl.

"Cyclolalkyl" is defined as a saturated cyclic aliphatic hydrocarboncontaining from at least 3 to as many as 8 carbon atoms. Preferredgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

"Aryl" is defined as a phenyl or naphthyl ring or a substituted phenylor naphthyl ring wherein one or more of the hydrogen atoms has beenreplaced by the same or different substituents as selected from R₁,COR₁, or R₂₅, where R₂₅ is defined as H, OH, halo, --OC(O)R₁₁,--C(O)R₁₁, --NR₁₁ C(O)R₁, NR₁₁ C(O)(CR₃ R₄)₂₋₆ R₁, --COOR₁₁, --COOR₁₁,--CONR₉,R₁₁, R₁₁ S--, --NR₉,R₁₁ SO₂ R₈, --SO₂ NR₉,R₁₁, nitro, cyano,--NR₁₁ CONR₉,R₁₁, where R₁,R₈, R₉,R₁₁, and R₁₂, are defined as above.

"Heteroaryl" is defined as an unsubstituted or an optionally substitutedmono- or bicyclic ring system of about 5 to about 12 carbon atoms andwhere each monocyclic ring may possess from 0 to about 4 heteroatoms,and each bicyclic ring my possess about 0 to about 5 heteroatomsselected from N, O, and S provided said heteroatoms are not vicinaloxygen and/or sulfur atoms and where the substituents, numbering from 0to about 5 may be located at any appropriate position of the ring systemand are optionally selected from the substituents listed for thosedescribed for aryl. Examples of such mono- and bicyclic ring systemswhich are by no means meant to limit the scope of this invention,including benzofuran, benzothiophene, indole, benzopyrazole, coumarin,isoquinoline, pyrrole, thiophene, furan, thiazole, imidazole, pyrazole,triazole, quinoline, pyrollidenone, pyrimidine, pyridine, pyridone,pyrazine, pyridazine, isothiazole, isoxazole and tetrazole.

"Aralkyl" refers to an alkyl group substituted by an aryl radical. Forexample, benzyl.

"Heteroaralkyl" refers to an alkyl group substituted by a heteroarylradical. For example, (4-pyridyl)methyl.

"Alkoxy" refers to an O-atom substituted by an alkyl, aryl or aralkyradical. For example methoxy, ethoxy, phenoxy, benzyloxy.

"Halo" means iodo, bromo, chloro, and fluoro.

The designation "(CR₃ R₄)₂₋₄ " refers to an alkyl linkage composed of atleast 2 but not more than 4 carbon atoms where said carbon atoms areindependently substituted with radicals described by R₃ and R₄. Examplesof such linkages include but are not limited to ethyl, propyl, butyl,2-methylethyl (--(MeHCCH₂ --), 2,2-dimethylethyl (Me₂ CCH₂ --).

The present invention also concerns the pharmaceutical composition andmethod of treatment of IL-1β protease mediated disease states ordisorders in a mammal in need of such treatment comprising theadministration of IL-1β protease inhibitors of formula (I) as the activeagent. These disease states and disorders include: infectious diseases,such as meningitis and salpingitis; septic shock, respiratory diseases;inflammatory conditions, such as arthritis, cholangitis, colitis,encephalitis, endocerolitis, hepatitis, pancreatitis and reperfusioninjury, immune-based disease, such as hypersensitivity; auto-immunediseases, such as multiple sclerosis; bone diseases; and certain tumorsand leukemias.

The present invention has particular utility in the modulation ofprocessing of IL-1β for the treatment of rheumatoid arthritis. Levels ofIL-1β are known to be elevated in the syncvial fluid of patients withthe disease. Additionally, IL-1β stimulates the synthesis of enzymesbelieved to be involved in inflammation, such as collagenase and PLA2,and produces joint destruction which is very similar to rheumatoidarthritis following intra-articular injection in animals.

In the practice of this invention an effective amount of a compound ofthe invention or a pharmaceutical composition thereof is administered tothe subject in need of, or desiring, such treatment. These compounds orcompositions may be administered by any of a variety of routes dependingupon the specific end use, including orally, parenterally (includingsubcutaneous, intraarticular, intramuscular and intravenousadministration), rectafly, buccally (including sublingually),transdermally or intranasally. The most suitable route in any given casewill depend upon the use, the particular active ingredient, and thesubject involved. The compound or composition may also be administeredby means of controlled-release, depot implant or injectable formulationsas described more fully herein.

In general, for the uses as described in the instant invention, it isexpedient to administer the active ingredient in amounts between about0.1 and 100 mg/kg body weight, most preferably from about 0.1 to 30mg/kg body weight for human therapy, the active ingredient will beadministered preferably in the range of from about 0.1 to about 20-50mg/kg/day: This administration may be accomplished by a singleadministration, by distribution over several applications or by slowrelease in order to achieve the most effective results. Whenadministered as a single dose, administration will most preferably be inthe range of from about 0.1 to mg/kg to about 10 mg/kg.

The exact dose and regimen for administration of these compounds andcompositions will necessarily be dependent upon the needs of theindividual subject being treated, the type of treatment, and the degreeof affliction or need. In general, parenteral administration requireslower dosage than other methods of administration which are moredependent upon absorption.

A further aspect of the present invention relates to pharmaceuticalcompositions comprising as an active ingredient a compound of thepresent invention in admixture with a pharmaceutically acceptable,non-toxic carrier. As mentioned above, such compositions may be preparedfor use for parenteral (subcutaneous, intraarticular, intramuscular orintravenous) administration, particularly in the form of liquidsolutions or suspensions; for oral or buccal administration,particularly in the form of tablets or capsules; or intranasally,particularly in the form of powders, nasal drops or aerosols.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived form fatty acids and ahexitol such as polyoxyethylene sorbitan monooleate, or condensationproducts of ethylene oxide with partial ester derived form fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample, ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxident such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative and flavoringand coloring agents.

When administered orally (or rectafly) the compounds will usually beformulated into a unit dosage form such as a tablet , capsule,suppository or cachet. Such formulations typically include a solid,semi-solid or liquid carrier or diluent. Exemplary diluents and vehiclesare lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma,aginates, tragacanth, gelatin, syrup, methylcellulose, polyoxyethylenesorbitar monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate,talc, and magnesium stearate.

Compositions for oral use may be prepared according to any method knownto the art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginin acid;binding agents, for example starch, gelatin or acacia, and lubricatingagents, for example magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed.

The compositions may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton,Pa., 1985. Formulations for parenteral administration may contain ascommon excipients sterile water or saline, alkylene glycols such aspropylene glycol, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, hydrogenated naphthalenes and the like. Examples ofvehicles for parenteral administration include water, aqueous vehiclessuch as saline, Ringer's solution, dextrose solution, and Hank'ssolution and nonaqueous vehicles such as fixed oils (such as corn,cottonseed, peanut, and sesame), ethyl oleate, and isopropyl myristate.Sterile saline is a preferred vehicle and the compounds are sufficientlywater soluble to be made up as a solution for all foreseeable needs. Thevehicle may contain minor amounts of additives such as substances thatenhance solubility, isotonicity, and chemical stability, e.g.,antioxidants, buffers, and preservatives. For oral administration, theformula can be enhanced by the addition of bile salts and also by theaddition of acylcarnitines (Am. J. Physiol. 251:332 (1986)).Formulations for nasal administration may be solid and contain asexcipients, for example, lactose or dextran, or may be aqueous or oilysolutions for administration in the form of nasal drops or meteredspray. For buccal administration typical excipients include sugars,calcium stearate, magnesium stearate, pregelatinated starch, and thelike.

When formulated for nasal administration the absorption across the nasalmucous membrane is enhanced by surfactant acids, such as for example,glycocholic acid, cholic acid, taurocholic acid, ethocholic acid,desoxycholic acid, chenodesoxycholic acid, dehydrocholic acid,glycodeoxy-cholic acid, and the like (See, B. H. Vickery, "LHRH and itsAnalogs-Contraception and Therapeutic Applications", Pt. 2, B. H.Vickery and J. S. Nester, Eds., MTP Press, Lancaster, UK, 1987).

DETAILED DESCRIPTION OF THE INVENTION

The compounds of this invention were prepared by using the generalsynthetic methods as described in Schemes 1, 2, 3, and 4. Z-Asparaticacid α-bromomethyl ketone (Scheme 1; Formula 1; Z=benzyloxyoarbonyl) istreated with an alcohol or a carboxylic acid in the presence of KF usingDMF as a solvent to give the α-substituted Z-aspartic acid methylketches (Formula 2). The preparation of bromide formula 1 and itsconversion to compounds of formula 2 is accomplished using the methodsas described by A. Krantz, et al. (Biochemistry, (1991 ), 30,4678-4687). Subsequently, the Z-group is removed to generate anN-terminal amine (Formula 3) under hydrogenolytic conditions. Thereagents and conditions typically used to carry out the hydrogenolyicremoval of the Z-group are hydrogen gas, ambient temperature andpressure, 5% palladium on carbon as the catalyst in an alcoholic solvente.g., methanol optionally containing two equivalent of hydrochloricacid. It is not necessary to purify the intermediate free amine (or thehydrochloride salt if hydrochloric acid is used in the hydrogenolysis),though this material needs to be dry and free of alcohol for thesubsequent coupling reaction to proceed in good yield. The amine(Formula 3) so obtained is then condensed with the pyrimidine carboxylicacid (Formula 4) to yield intermediates of Formula 5. It is generallynecessary to first activate the pyrimidine carboxylic acid as an acidchloride or mixed anhydride and then react it with the free amine (orhydrochloride salt) in the presence of an organic base, e.g.,N-methylmorpholine. Alternatively, coupling the pyrimidine carboxylicacid with the intermediate amine is conducted using amide couplingreagents/conditions employed in peptide coupling chemistry ("ThePractice of Peptide Synthesis." M. Bodanszky, Springer-Verlag, N.Y.,1984; The Peptides. Vol 1-3, E. Gross and J. Meienhofer, Eds. AcademicPress, N.Y., 1981 ). The remaining synthetic transformation to generatethe ICE inhibitors is the hydrolysis of the t-butyl ester function. Thisis conducted by exposing the t-butyl ester (Formula 5) to a 25% solutionof trifluoroacetic acid (TFA) in methylene chloride at 25° C. Thede-esterification is usually complete with 3 h. Removal of the volatileTFA and organic solvent affords the aspartic acid (Formula 6). The yieldof the reaction is quantitative in most instances, providing the t-butylester starting material is of high purity. Purification, if required,can be performed by recrystallization or chromatographic techniqueswhich are well know to those skilled in the art. The concentration ofTFA may range from 5%-100% and other organic solvents may be used suchas chloroform. Also, a solution of three molar anhydrous hydrochloricacid in ethyl acetate may be used in place of the TFA-methylene chloridesolution with equal efficiency.

Scheme 2 outlines the synthesis of the aspartyl aldehyde containingpyrimidines. The starting material for their synthesis is the aspartylsemicarbazone (Formula 7). The Z-group is removed via standardhydrogenation conditions to yield the corresponding amine (Formula 8).This is then coupled to the pyrimidine acid (Formula 4) using couplingconditions analogous to those described above. A double de-protection isrequired to free the beta carboxylic acid (trifluoracetic acid) and thealfa aldehyde (37% aqueous paraformaldehyde, acetic acid, methanol)yielding compounds of Formula 10.

Scheme 3 outlines an alternate synthetic method for introducing R₁groups onto the pyrimidine 5-amino function further enhancing the scopeof this invention. Pyrimidines either as their free acids, esters oraspartic acid amides which contain a Z-group (Formula 11) may besubjected to hydrogenolysis conditions (similar to those describedabove) to yield the corresponding 5-amino pyrimidines (Formula 12). Theamine moiety may be reacted with acid chlorides, or activated carboxylicacids (conditions analogous to those used to couple Formula 3 and 4 asdescribed in Scheme 1 above) to afford R₁ containing pyrimidines withstructural diversity in R₁.

Scheme 4 outlines the synthesis of the requisite pyrimidines. Thestarting materials used are the 3-carboxyethyl pyrimidines with eitherthe N-ally (Formula 13) or N-acetaldehyde dimethyl acetal (Formula 14).Their synthesis can be readily deduced by those in the art employing thereaction conditions presented in the literature (Veale, C. A.; et al, J.Org. Chem. (1993), 58, 4490-4493; Gupta, K. A.; et al. Ind. J. Chem. B,21B, 228; Nemeryuk, M. P.; et al. Collect. Czech. Chem Commun. (1986),51,215-233). The ethyl esters are hydrolyzed in the presence of aqueousbase (LiOH in H₂ O-THF or NaOH in H₂ O-THF) to give the correspondingacids (Formulas 15 and 16). The carboxylic acids in turn are subjectedto a Curtius rearrangement (Pfister, J. R.; et al. Synthesis, (1983),38; Radhakrishna, A. S.; et al. Synthesis, (1983), 538; Ninomiya, K.; etal; Tetrahedron, (1974), 30, 2151) yielding a highly reactive isocyanate(Formula 17 and 18), which is not isolated, but reacted immediately withan alcohol or an amine (see Ninomiya, K. et al. Supra). The overallprocess provides either a carbamate (isocyanate trapped with an alcohol)or a urea (isocyanate trapped with an amine), as represented by Formulas19 and 20. At this point the synthesis diverges in that if an N-allylpyrimidine was used a starting material (Formula 19), the olefin isoxidized with osmium tetroxide/N-methyl morphofine N-oxide (See, V.VanRheenen; et al. Tetrahedron Lett., (1976), 1973-1976; OrganicSynthesis Vol 58, p43-51) and sodium or potassium periodate (H. O.House; Modern Synthetic Reactions, W. A. Benjamin Inc., Menlo Park,Calif. 1972,353-359) yielding the intermediate aldehyde (Formula13→Formula 19→Formula 21). Alternatively, if an N-acetaldehyde dimethylacetal was used as a starting material (Formula 20), the dimethylacetalfunctionality is treated with dilute acid (aqueous HC1 ) liberatingintermediate aldehyde (Formula 14→Formula 20→Formula 21). Acids offormula 4 were obtained from aldehydes of formula 21 via sodium orpotassium chlorite-mediated oxidation (B. S. Bal; et al. Tetrahedron,(1981 ), 37, 2091 ). It should be noted that trapping the isocyanate(formulas 17 and 18) with benzyl alcohol provides intermediateZ-carbamates which ultimately lead to the compound of formula 11 (Scheme3). ##STR9## wherein:

R₁ -R₄, R₉, R₁₁, and R₂₄ are as defined in formula (I), Z is defined asthe benzyloxycarbonyl group, W is defined as an OH group, a HNC(H)(CH₂OOtBu)COCH₂ R₂₆) and a HNC(H)(CH₂ COOtBu)C═NNHCONH₂ moieties, where R₂₆is defined as F, --O(CO)₀₋₁ -aryl,--OP(O)(R₁₄)R.sub.(15), ##STR10##wherein R₈, R₁₄, R₁₅, R₁₆, R₁₇ and R₁₈ are defined as previously.

The following will further illustrate the compounds of the presentinvention.

EXAMPLE 1 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl-L-aspartic acid 2,6-dichlorobenzoyloxymethyl ketone

Part A: N-Benzyloxycarbonyl-L-aspartic acid bromomethyl ketoneβ-tert-butyl ester (0.3 g; 0.76 mM) was dissolved in 12 mL of anyhydrousDMF. To this solution was added powdered potassium fluoride (0.11 g; 19mmol) and 2,6-dichlorobenzoic acid (0.17 g; 0.91 mmol) and the reactionmixture was stirred overnight. The solution was diluted with Et₂ O andwashed with water, aqueous saturated NaHCO₃, brine and dried (MgSO₄).The ketone so obtained was purified by silica gel chromatography usingethyl acetate/hexane as the eluting solvent (¹ H NMR (CDCl₃) §7.36 (m,9H), 5.90 (d, 1H), 5.20 (m, 4H), 4.67 (m, 1H), 3.00 and 2.75 (doublet ofdoublets, 1H each), 1.42 (s, 9H)).

Part B: N-Benzyloxycarbonyl-L-aspartic acid 2,6-dichlorobenzoyloxymethylketone β-tert-butyl ester (1.02 g, 2 mmol; Part A above) was dissolvedin absolute ethanol (100 mL, 4 mmol) containing 2 equiv. of 6N aqueousHCl (4 mmol) and 10% palladium on carbon (96 mg). The reaction mixturewas stirred under an ambient atmosphere of hydrogen gas forapproximately I hour (thin layer chromatography 5% MeOH--CH₂ Cl₂ !indicated the disappearance of starting material). The solution wasfiltered and the solvent was removed in vacuo to give L-aspartic acid2,6-dichlorobenzoyloxymethyl ketone β-tert-butyl ester HCl salt whichwas used immediately in the subsequent reaction described in Part C.

Part C: A solution of2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl) acetic acid (771 mg, 2.05 mmol) in CH₂ Cl₂ (10mL) was cooled to -20° C. and isobutylchloroformate (0.28 mL, 2.05 mmol)and N-methylmorpholine (0.23 mL, 2.05 mmol) were added sequentially. Thereaction mixture was stirred for 15 minutes and a solution of asparticacid 2,6-dichlorobenzoyloxy methyl ketone β-tert-butyl ester HCl salt(prepared in Part B above) was added followed by a second addition ofN-methyl morpholine (0.23 mL, 2.05 mmol). The reaction mixture wasstirred for 30 minutes and then was diluted with EtOAc, washed withwater, aqueous saturated NaHCO₃, brine and dried (MgSO₄). The solventswere removed in vacuo and the product purified by silica gelchromatography using 40% EtOAc-hexane as eluent to give N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl-)1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid2,6-dichlorobenzoyloxymethyl ketone β-tert-butyl ester (1.2 g; 80%).

Part D: A solution of N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid2,6-dichlorobenzoyloxymethyl ketone β-tert-butyl ester (Part C above) inmethylene chloride containing 25% v/v trifluoroacetic acid (20 mL) wasstirred for 2 hours at 0° C. The solvent was removed in vacuo and theresidue was purified by silica gel chromatography to give analyticallypure N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 2,6-dichlorobenzoyloxymethyl ketone lowresolution mass spectrum: m/z=699 (M+H).

EXAMPLE 2

N-2-(5-Thiomethylbenzoylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihyro-1-pyrimidinyl)acetoyl!L-aspartic acid 2,6-dichlorobenzoyloxymethyl ketone

Part A: N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pydmidinyl)acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazoloxymethyl ketoneβ-tert-butyl ester (2.5 g, 3.0 mmol) was dissolved in absolute ethanol(100 mL, 4 mmol) containing 2 equiv of 6N aqueous HCl. The solution wasdegassed with nitrogen and 10% palladium on carbon was added (300 mg).The reaction mixture was stirred under an ambient atmosphere of hydrogengas for approximately 5 h (thin layer chromatography 50% EtOAc--hexane:Rf starting material=0.5; R_(f) of product=0.0! indicated thedisappearance of starting material). The solution was filtered and thesolvent was removed in vacuo to give N-2-(5-amino-6-oxo-2-(4-fluorophenyl-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl) pyrazoloxymethyl ketoneβ-tert-butyl ester which was azeotroped with toluene and used withoutfurther purification in the subsequent reaction described in Part B.

Part B: To a solution of N- 2-(5-amino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)-acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazoloxymethyl ketoneβ-tert-butyl ester (713 mg, 1.0 mmol prepared in Part A above) inmethylene chloride (30 mL) at 5° C. was added 4-thiomethylbenzoylchloride (279 mg, 1.5 mmol) followed by the addition ofN-methylmorpholine (0.5 mL; 4.5 mmol) and 4-N,N-dimethylaminopyridine(10 mg). The reaction mixture was stirred for 2 h at 5° C. and then wasallowed to warm to room temperature. The solution was diluted withEtOAc, washed with water, saturated aqueous NaHCO₃, brine and dried(MgSO₄). The solvents were removed in vacuo. The product was purified bysilica gel chromatography using about 30% EtOAc-hexane as eluent to giveN-2-(5-(4-thiomethylbenzoylamino)-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazoloxymethyl ketoneβ-tert-butyl ester in 50% yield.

Part C: N- 2-(5-(4-Thiomethylbenzoylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl) pyrazoloxymethyl ketoneβ-tert-butyl ester was converted to its corresponding acid, N-2-(5-(4-thiobenzoylamino)-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazoloxymethyl ketone usingthe conditions described in Part D of example 1 above.

Mass spectrum: m/z=787 (M+H)

Following the procedure in Schemes 1 and 2, and by analogy with Examples1 and 2, the following compounds were prepared.

EXAMPLE 3 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid diphenylphosphinoxymethyl ketone

Mass spectrum: m/z=727 (M+H)

EXAMPLE 4 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazoloxymethyl ketone

Mass spectrum: m/z=771 (M+H)

EXAMPLE 5 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(3-pheny)coumarinyloxymethyl ketone

Mass spectrum: m/z=747 (M+H)

EXAMPLE 6 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone

Mass spectrum: m/z=737 (M+H)

EXAMPLE 7 N-2-(5-Isopropyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone

Mass spectrum: m/z=671 (M+H)

EXAMPLE 8 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(3-pyridinyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone

Mass spectrum: m/z=720 (M+H)

EXAMPLE 9 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1.6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone

Mass spectrum: m/z=725 (M+H)

EXAMPLE 10 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-methyl-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-asparticacid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethyl ketone

Mass spectrum: m/z=657 (M+H)

EXAMPLE 11 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(2-pyridinyl)-3-trifluoromethyl) pyrazoloxymethyl ketone

Mass spectrum: m/z=726 (M+H)

EXAMPLE 12 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl-L-asparticacid 5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazoloxymethyl ketone

Mass spectrum: m/z=759 (M+H)

EXAMPLE 13 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl-L-asparticacid 2,6-dichlorobenzoyloxymethyl ketone

Mass spectrum: m/z=687 (M+H)

EXAMPLE 14 N-2-(5-Benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid aldehyde

Mass spectrum: m/z=485 (M+H)

Compounds of the present invention were tested for IL-1β proteaseinhibition activity according to the following protocols:

In Vitro

Second order rate constants for inactivation were obtained using theenzyme assay described in Dolle, R. E. et al.; J. Medicinal Chemistry,(1994), 37, 781.

The compounds in examples 1-13 possess IL-1β protease inhibition (kobs/Iwere>50,000 M⁻¹ s⁻¹).

In Vivo

In vivo inhibition (IC₅₀) was determined based on Miller et al.,"Inhibition of Mature" IL-1β Production in Murine Macrophages and aMurine Model of Inflammation By WIN 67694, An Inhibitor of IL-1βConverting Enzyme, J. Immunol., 1994, 154, 1331, as follows:

Human monocytes were isolated from heparinized leukopheresis unitsobtained through Biological Specialty Corporation (Lansdale, Pa.).Monocytes were purified by Ficoll-Hupaque (Pharmacia Fine Chemicals,Piscataway, N.J.) gradient centrifugation and more than 95% puremonocyte populations obtained by centrifugal elutriation. The assay wasperformed on duplicate samples of freshly isolated human monocytes,cultured in suspension at 37° C. and rotated gently in conical bottompolypropylene tubes (Sardstedt Inc., Princeton, N.J.). Human monocytesat a concentration of 5×10⁶ cells/mL were resuspended in 1 mL of RPMI1640 (a common tissue buffer from M. A. Bioproducts, Walkersville, Md.)containing 1% fetal calf serum (FCS) (HyClone, Logan, UT) and 50 μg/mLgentamycin (Gibco, Grand Island, N.Y.). The cells were treated eitherwith a compound of the invention (i.e. test compound) or with anon-inhibitor (control compound, typically 0.03% DMSO) for 15 minutesand then activated with 0.01% fixed Staphylococcus aureus (The EnzymeCenter, Malden, Mass.) for 1 hour. The cells were then centrifuged andresuspended in 1 mL of cysteine, methionine-free RPMI media containing1% dialyzed FCS (Hyclone). The cells were pretreated with a testcompound or control compound for 15 minutes after which 0.01% fixed S.aureus plus 100 μCi Tran 35-S label (ICN, Irvine, Calif.) was added andthe cells incubated at 37° C. for 1 hour. After incubation, mL RPMIcontaining 1% fetal calf serum. The cells were again pretreated with atest or control compound for 15 minutes and then 0.01% S. aureus for 2hours. At the end of the incubation, cells were centrifuged andsupernates saved for immunoprecipitation. Cells were washed once inphosphate buffer saline and then lysed in RIPA, a continuous cell mediabuffer containing 2 mM phenylmethylsulfonyl fluoride, 10 mM iodoacetate,1 μg/mL pepstatin A, 1 μg/mL leupeptin and 0.5 TIU aprotinin.

For the immunoprecipitations, an equal volume of 1% dry milk in RIPAbuffer plus 50 μL of resuspended protein A sepharose CL-4B (Pharmacia,Piscataway, N.Y.) was added to supernates and 1 mL of 4% dry milkcontaining protein A sepharose CL-4B to cell lysates and samples rotatedfor 30 minutes at 4° C. Beads were then centrifuged down, samplestransferred to fresh tubes and incubated overnight with 40 μg rabbitanti-human IL-1β polyclonal antibody (Genzyme, Cambridge, Mass.). TheIL-1β proteins were then precipitated with 70 μL protein A sepharose,resuspended in 60 μL SDS sample buffer and run on 15% SGD-PAGE gels.Autoradiography was performed on dried gels and the amount ofradioactivity (counts per minute, cpm) quantitated using a Betascope 603analyzer.

Data Analysis

In the monocyte pulse chase assay, each test parameter was run induplicate. Data was collected from the Beta Scope using a personalcomputer, then transferred to the VAX system for calculation of mean cpmand standard deviation of the mean. When test compounds were evaluated,the percent inhibition of release of mature IL-1β was calculated asfollows:

100× 1-(cells treated with stimuli+test compound-unstimulatedcells)/(cells treated with stimuli+control compound-unstimulated cells)!

These % inhibition values were then used to calculate IC₅₀ value foreach compound. Since the human monocyte pulse chase assay uses primarycells from different donors, each test compound was run in 2-3 separateexperiments, using monocytes from 2-3 different donors.

For examples 1,6,7 and 9, the in vivo IC₅₀ 's ranged from approximately0.1 to up to approximately 10 μM.

Elastase Inhibition (In Vitro)

Compounds of examples 1, 6 and 7 were examined for their ability toinhibit elastase. The in vitro assay was carried out as described byCha, Biochem. Pharmacol., (1975), 24, 2177-2185. Examples 1,6 and 7which are representative of this class of ICE inhibitor, did not inhibitelastase with IC₅₀ 's≧10 μM.

Having described the invention with reference to its preferredembodiments, it is to be understood that modifications within the scopeof the invention will be apparent to those skilled in the art.

What is claimed is:
 1. A compound selected from the group consistingof:N- 2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl-acetoyl!-L-aspartic acid2,6-dichlorobenzoyloxymethyl ketone; N-2-(5-thiomethyl-benzoylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihyro-1-pyrirnidinyl) acetoyl!-L-aspartic acid2,6-dichlorobenzoyloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic aciddiphenylphosphinoxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl-1, 6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl) pyrazoloxymethyl ketone N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(3-pheny)coumarinyloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-phenyl-3-fluoromethyl)-pyrazoloxymethyl ketone; N-2-(5-isopropyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone; N- 2-(5-benzyloxycarbonylamino-6-oxo-2-(3-pyridinyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone; N- 2-(5-benzyloxycarbonylamino-6-oxo-2-methyl-1,6-dihydro-1-pyrimidinyl)-acetoyl!-L-aspartic acid5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(2-pyridinyl)-3-trifluoromethyl)pyrazoloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl-3-trifluoromethyl)pyrazoloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 2,6-dichlorobenzoyloxymethyl ketone; and N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid aldehyde.
 2. A pharmaceutical composition forinhibiting interleukin-1β protease comprising a compound selected fromthe group consisting of:N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 2,6-dichlorobenzoyloxymethyl ketone; N-2-(5-thiomethylbenzoylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 2,6-dichlorobenzoyloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid diphenylphosphinoxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazoloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)-acetoyl!-L-aspartic acid5-(3-pheny)coumarinyloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(4-fluorophenyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone; N-2-(5-isopropyloxycarbonylamino-6-oxo-2-phenyl-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(3-pyridinyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-methyl-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid 5-(1-phenyl-3-trifluoromethyl)pyrazoloxymethylketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid5-(1-(2-pyridinyl)-3-trifluoromethyl)pyrazoloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl-L-aspartic acid5-(1-(4-chlorophenyl)-3-trifluoromethyl)pyrazoloxymethyl ketone; N-2-(5-benzyloxycarbonylamino-6-oxo-2-(2-thienyl)-L-aspartic acid 2,6-dichlorobenzoyloxymethyl ketone; and N-2-(5-benzyloxycarbonyl-amino-6-oxo-2-(2-thienyl)-1,6-dihydro-1-pyrimidinyl)acetoyl!-L-aspartic acid aldehyde.